Retroviral vectors derived from C-type murine leukaemia viruses (MLVs) have emerged as highly versatile gene delivery vehicles and have been selected for use in many human gene therapy protocols, especially those requiring transduction of normal or neoplastic haemopoietic cells. In the interests of safety and efficacy, particularly for direct genetic modification of target cells in vivo, it is desirable that retroviral gene delivery should be accurate but the clinically approved (amphotropic) retroviral vectors in current use attach to an ubiquitously expressed cellular receptor and therefore infect human cells promiscuously. Cell-selective retroviral gene delivery might be achieved by modification of the viral membrane spike glycoproteins responsible for receptor-mediated virus entry.
Retroviral envelope glycoproteins mediate specific viral attachment to cell surface receptors and subsequently trigger fusion between the viral envelope and the target cell membrane. Retroviral envelope glycoproteins consist of an external glycoprotein moiety (SU) noncovalently attached at its C-terminus to a smaller transmembrane polypeptide moiety (TM). Each surface projection (or spike), visible by electron microscopy on the viral surface is a trimer of identical envelope glycoprotein subunits. SU comprises two domains connected by a proline-rich hinge, the N-terminal domain conferring receptor specificity and exhibiting a high degree of conservation between MLV's with different host ranges (Battini et al. 1992 J Virol 66 p1468-1475).
The crystallisation of retroviral SU proteins or their domains remains elusive but sequence alignment of the SU receptor-binding domains of C-type retroviruses reveals a high degree of homology, presumably reflecting a conserved 3-dimensional fold. Two discontinuous hypervariable peptide sequences in this domain, VRA and VRB, have been identified as major determinants of receptor-binding specificity (Battini et al, 1995 J Virol 69 p713-719). Thus it is probable that these receptor binding domains consist of a conserved framework acting as a scaffold for presentation of hypervariable peptide loops which confer binding specificity. According to the "canyon hypothesis", the receptor binding site in SU is likely to take the form of a pocket or groove whose walls and floor may be formed by the residues in VRA and VRB.
Retroviral host range is determined in part by the species and tissue distribution of specific cell surface receptors that are recognised by the viral envelope glycoprotein. Moloney MLV is an ecotropic virus whose envelope attaches to mouse and rat cells but not to human cells. 4070A MLV is an amphotropic virus whose envelope attaches to cells of mouse and human origin. Three C-type retrovirus receptors have now been identified and all are membrane permeases with multiple membrane-spanning domains. In the case of Moloney MLV, the precise target for virus attachment is a constrained peptide loot in the third extracellular domain of the murine cationic amino acid transporter (CAT1) which also functions as a Moloney virus receptor when transplanted into the corresponding site on a homologous human protein (Albritton et al, 1993 J Virol 67 p2091-2096).
Naturally occurring retroviruses incorporate a single species of envelope glycoprotein which mediates specific high-affinity binding to a single well-defined cognate cell-surface receptor. However, additional receptor-binding domains can be incorporated to generate retroviral particles that are capable of binding to more than one species of cellular receptor. Thus, with dual viral infection of a single cell by two enveloped viruses, the host range of either virus may be predictably extended due to promiscuous incorporation of spike glycoproteins encoded by both viruses (Levy, 1976 Virology 77 p811-825; Weiss & Wong, 1977 Virology 76 p826-834; Besmer & Baltimore, 1977 J Virol 21 p965-973; Canivet et al. 1990 Virology 178 p543-541; Lusso et al 1990 Science 247 p848-852: Spector et al. 1990 J Virol 64 p2298-2308; Schnitzer et al, 1977 J Virol 23 p449-454: Metsikko & Garoff, 1989 J Virol 63 p5111-5118; Schubert et al, 1992 J Virol 66 p1579-1589).
Non-viral polypeptide ligands can also be co-incorporated (or pseudotyped) into retroviral vectors with unmodified retroviral envelope glycoproteins. Thus, MHC antigens are incorporated into the envelopes of human and simian immunodeficiency viruses (Gelderblom et al, 1987 Z Naturforsch 42 p1328-1334; Schols et al, 1992 Virology 189 p374-376) mammalian CD4 expressed in avian (quail) cells was incorporated into the envelopes of budding avian retroviruses (Young et al, 1990 Science 250 p1421-1423), a CD4-envelope chimaera, retaining the membrane anchor sequence of the retroviral envelope glycoprotein, was co-incorporated into avian retroviruses with wild type avian retroviral envelope glycoproteins (Young et al. 1990 Science 250 p1421-1423) and a single chain antibody/envelope chimaera was similarly co-incorporated into avian retroviruses (Chu & Dornberg, 1995 J Virol 69 p2659-2663). In the latter example, the host range of the retroviruses was extended to otherwise non-permissive cells that express the tumour antigen recognised by the displayed single chain antibody. A chimaeric Moloney MLV envelope glycoprotein in which the N-terminal domain of SU had been substituted by erythropoietin was co-incorporated into MLV particles with unmodified Moloney envelope glycoprotein (Kasahara et al, 1994 Science 266 )1373-1376). In this example, the authors claimed that the host range of the erythropoietin-displaying viruses was extended to human cells expressing the erythropoietin receptor and that their infectivity was enhanced on mouse fibroblasts that had been transfected with an expression vector coding for erythropoietin receptor. Non-viral polypeptide ligands have also been displayed on retroviruses as N-terminal extensions of a substantially intact Moloney MLV retroviral envelope glycoprotein which retains its receptor-binding domain (Russell et al, 1993 NAR 21 p1081-1085).
In summary, several non-viral polypeptide ligands have been co-incorporated into retroviral vectors with unmodified envelope proteins. The purpose of these manipulations has been to selectively enhance the infectivity of the recombinant retroviruses for cells expressing receptors that are recognised by the non-viral ligand. Selective inhibition of retroviral entry into cells expressing receptors for a displayed non-viral ligand has not previously been proposed, observed or discussed.